Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

The present invention provides compositions and methods for targeting
polypeptides to the chloroplasts of higher plants. Compositions include
expression cassettes having a nucleotide sequence encoding a chloroplast
targeting peptide (CTP) operably linked to a nucleotide sequence of
interest, wherein the CTP is derived from Chlamydomonas sp. Plant
transformation vectors, plants and plant cells having the CTP sequences
are also encompassed, as well as variants and fragments of the CTP
sequences. Methods for expressing a heterologous nucleotide sequence in a
plant using the CTP sequences disclosed herein are also provided.

Claims:

1. A plant cell having stably incorporated into its genome an expression
cassette comprising a nucleotide sequence encoding a chloroplast
targeting peptide (CTP), wherein said nucleotide sequence encoding said
CTP is operably linked to a nucleotide sequence of interest, and wherein
said nucleotide sequence encoding said CTP is selected from the group
consisting of: a) the nucleotide sequence set forth in SEQ ID NO:1 or 2;
b) a nucleotide sequence encoding the amino acid sequence set forth in
SEQ ID NO:3; and, c) a nucleotide sequence encoding an amino acid
sequence having at least 90% sequence identity to SEQ ID NO:3, wherein
said amino acid sequence is a chloroplast transit peptide.

2. The plant cell of claim 1, wherein said plant cell is from a monocot.

3. The plant cell of claim 1, wherein said plant cell is from a dicot.

4. A plant comprising the plant cell of claim 1.

5. The plant of claim 4, wherein said plant is a monocot.

6. The plant of claim 4, wherein said plant is a dicot.

7. A seed derived from the plant of claim 7, wherein said seed comprises
the nucleotide sequence encoding said chloroplast transit peptide.

9. A method for expressing a nucleotide sequence of interest in a plant,
said method comprising: a) introducing into a plant cell an expression
cassette comprising a nucleotide sequence encoding a chloroplast
targeting peptide (CTP), wherein said nucleotide sequence encoding said
CTP is operably linked to said nucleotide sequence of interest, and
wherein said nucleotide sequence encoding said CTP is selected from the
group consisting of i) the nucleotide sequence set forth in SEQ ID NO:1
or 2; ii) a nucleotide sequence encoding the amino acid sequence set
forth in SEQ ID NO:3; and, iii) a nucleotide sequence encoding an amino
acid sequence having at least 90% sequence identity to SEQ ID NO:3,
wherein said amino acid sequence is a chloroplast transit peptide; and,
b) regenerating a transformed plant from said plant cell; wherein said
plant has stably incorporated into its genome said expression cassette.

10. The method of claim 9, wherein said plant is a dicot.

11. The method of claim 9, wherein said plant is a monocot.

12. The method of claim 9, wherein said nucleotide sequence of interest
encodes a gene product that confers herbicide or pest resistance.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser.
No. 12/555,175, filed Sep. 8, 2009, which claims the benefit of U.S.
Provisional Patent Application No. 61/095,134, filed Sep. 8, 2008, which
is hereby incorporated in its entirety by reference herein.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The official copy of the sequence listing is submitted
electronically via EFS-Web as an ASCII formatted sequence listing with a
file named "APA056USO 1DSEQLIST.txt", created on Mar. 22, 2013, and
having a size of 34 kilobytes and is filed concurrently with the
specification. The sequence listing contained in this ASCII formatted
document is part of the specification and is herein incorporated by
reference in its entirety.

FIELD OF THE INVENTION

[0003] The present invention relates to the field of plant molecular
biology, more particularly to the identification and use of regulatory
elements in plants.

BACKGROUND OF THE INVENTION

[0004] Chloroplast biogenesis in plants is dependent upon the coordinated
activities of two independent genetic systems localized in the
chloroplast and the nucleus (see Cline and Henry (1996), Annu. Rev. Cell
Dev. Biol. 12, 1-26). The vast constituent chloroplast proteins are
encoded by the nuclear genes and are synthesized cytoplasmically--as
precursor forms which contain N-terminal extensions known as transit
peptides. The transit peptide is instrumental for specific recognition of
the chloroplast surface and in mediating the post-translational
translocation of pre-proteins across the chloroplast envelope and thence
to the various different subcompartments within the chloroplast (e.g.
stroma, thylakoid and thylakoid membrane).

[0006] Compositions and methods for chloroplast targeting of polypeptides
in a plant are provided. Compositions comprise expression cassettes
comprising a nucleotide sequence encoding a chloroplast targeting peptide
(or chloroplast transit peptide, "CTP") sequence derived from an algal
organism operably linked to nucleotide sequence of interest. These
expression constructs are useful for expression and proper targeting of
the nucleotide sequence of interest in a monocot or a dicot plant. The
invention further provides vectors comprising the expression cassettes,
and plants and plant cells having stably incorporated or transiently
expressed into their genomes an expression cassette described above.
Additionally, compositions include transgenic seed of such plants.

[0007] Methods are also provided for expressing a nucleotide sequence in a
plant or plant cell, as well as methods for identifying algal CTP
sequences for use in a plant.

[0010] FIG. 3 demonstrates the calculation of the molecular weight of the
processed Chlamydomonas EPSPS--GRG-23(ace3)(R173K) protein expressed in
maize. A linear regression of the plot of Log Molecular Weight vs.
Distance Migration of the protein molecular weight standards from FIG. 2
was used to calculate the apparent molecular weight of the
GRG23(ace3)(R173K) protein standard and the processed Chlamydomonas
EPSPS--GRG-23(ace3)(R173K) detected in plant extract.

DETAILED DESCRIPTION

[0011] In the production of transgenic plants it is often useful to direct
foreign proteins to specific subcellular locations, e.g., the
chloroplast, vacuole, mitochondria, or ER. Previous workers have fused
DNA sequences encoding transit peptides from various plant genes to the
genes of interest. When the gene is translated the resulting protein has
the plant transit peptide fused to the amino terminus of the protein of
interest, and thus the protein is directed, with varying efficiency, to
the desired subcellular compartment.

[0012] Thus, the present invention is drawn to compositions and methods
for chloroplast targeting of polypeptides in higher plants or plant
cells. The compositions of the present invention comprise expression
cassettes comprising a nucleotide sequence encoding a chloroplast transit
peptide (CTP) derived from an algal organism operably linked to a
nucleotide sequence of interest. In one embodiment, the CTP is derived
from Chlamydomonas sp. In another embodiment, the CTP comprises the amino
acid sequence set forth in SEQ ID NO:3, 5, or 7 or an amino acid sequence
encoded by SEQ ID NO:1, 2, 4, or 6, as well as variants, fragments, and
derivatives thereof. In addition, transformed plants, plant cells, and
seeds are provided.

[0013] The CTP-encoding sequences of the invention, when assembled within
a DNA construct such that the CTP-encoding sequence is operably linked to
a nucleotide sequence of interest, facilitate co-translational or
post-translational transport of the peptide of interest to the
chloroplast of a plant cell stably transformed with this DNA construct.
Methods for expressing a nucleotide sequence in a plant comprise
introducing into plant cells an expression cassette comprising a
CTP-encoding nucleotide sequence of the invention operably-linked to a
nucleotide sequence of interest, and regenerating a transformed plant
from the plant cell.

[0014] The articles "a" and "an" are used herein to refer to one or more
than one (i.e., to at least one) of the grammatical object of the
article. By way of example, "an element" means one or more elements.

[0015] As used herein, the term "nucleic acid molecule" is intended to
include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules
(e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide
analogs. The nucleic acid molecule can be single-stranded or
double-stranded, but preferably is double-stranded DNA.

Chloroplast Transit Peptides

[0016] Chloroplasts are organelles found in plant cells and eukaryotic
algae that conduct photosynthesis. The chloroplast is a complex cellular
organelle composed of three membranes: the inner envelope membrane, the
outer envelope membrane, and the thylakoid membrane. The membranes
together enclose three aqueous compartments termed the intermediate
space, the stroma, and the thylakoid lumen. While chloroplasts contain
their own circular genome, many constituent chloroplast proteins are
encoded by the nuclear genes and are cytoplasmically-synthesized as
precursor forms which contain N-terminal extensions known as chloroplast
transit peptides (CTPs). The CTP is instrumental for specific recognition
of the chloroplast surface and in mediating the post-translational
translocation of pre-proteins across the chloroplast envelope and into
the various different subcompartments within the chloroplast (e.g.
stroma, thylakoid and thylakoid membrane).

[0017] At least two distinct functional domains have been identified in
chloroplast transit peptides: the stromal targeting domain (STD) and the
lumen targeting domain (LTD). STDs govern access to the general import
pathway and are both necessary and sufficient for import of the passenger
protein to the stroma. Stromal protein precursors possess transit
peptides that contain only an STD, whereas thylakoid lumenal protein
precursors have both an STD and an LTD.

[0018] STDs range in length from about 30 to 120 residues and are rich in
hydroxylated residues and deficient in acidic residues. They tend to
share several compositional motifs: an amino terminal 10-15 residues
devoid of Gly, Pro and charged residues; a variable middle region rich in
Ser, Thr, Lys and Arg; and a carboxy-proximal region with loosely
conserved sequence (Ile/Val-X-Ala/Cys-Ala; SEQ ID NO:17) for proteolytic
processing. However, there are no extensive blocks of sequence
conservation, nor any conserved secondary structural motifs. Theoretical
analyses suggest that STDs adopt predominantly random coil conformations.

[0020] The present invention discloses the use of CTPs derived from algal
species, particularly Chlamydomonas sp., in higher plants. For the
purposes of the present invention, "higher plants" are considered members
of the subkingdom Embryophytae. In one embodiment, the CTP useful in the
methods and compositions disclosed herein is derived from Chlamydomonas.
In another embodiment, the CTP is set forth in SEQ ID NO:3, 5, or 7, or
is encoded by SEQ ID NO:1, 2, 4, or 6, including variants, fragments, and
derivatives thereof. However, one of skill in the art would understand
how to identify chloroplast transit peptides other than the ones
disclosed herein. For example, a number of CTPs (or protein sequences
comprising CTPs) are listed in GENBANK®.

[0021] The CTPs disclosed herein are useful for targeting a polypeptide to
the chloroplast of a plant cell. In one embodiment, the CTPs disclosed
herein provide improved translocation compared to CTPs derived from, for
example, higher plant organisms. The CTPs disclosed herein may result in
an at least about 20%, at least about 30%, at least about 40%, at least
about 50%, at least about 60%, at least about 70%, at least about 80%, at
least about 90%, at least about 100%, or greater, or at least about
2-fold, at least about 3-fold, at least about 4-fold, or greater
improvement in translocation of the polypeptide into the chloroplast when
compared to a reference CTP. An improvement can be measured in terms of
the amount of polypeptide that gets translocated into the chloroplast,
the amount of active polypeptide that gets translocated into the
chloroplast, or both. An improvement can also be measured in terms of an
improvement in the phenotype of an organism transformed with the
chloroplast-targeted protein of interest. For example, where the CTP of
the invention is used to target an herbicide resistance protein to the
chloroplast of the plant, an improvement in activity can be measured in
terms of an improvement in herbicide resistance.

Expression Cassettes

[0022] The CTP-encoding sequences of the invention may be provided in an
expression cassette that allows it to drive expression and localization
of a polypeptide encoded by the nucleotide sequence of interest into the
chloroplast of plant cells. By "expression cassette" is intended a DNA
construct that is capable of resulting in the expression of a protein
from an open reading frame in a cell. The cassette will include in the
5'-3' direction of transcription, a transcriptional initiation region
preferably comprising a promoter suitable for expression in a plant cell
of interest, operably-linked to a CTP-encoding sequence of the invention,
which is further operably linked to a nucleotide sequence of interest,
and a translational and transcriptional termination region (i.e.,
termination region) functional in plants. The CTP-encoding nucleotide
sequence and the nucleotide sequence of interest may be separated from
one another by nucleotide sequences encoding one or more "linker" amino
acids as discussed elsewhere herein.

[0023] The cassette may additionally contain at least one additional gene
to be cotransformed into the organism, such as a selectable marker gene.
Alternatively, the additional gene(s) can be provided on multiple
expression cassettes. Such an expression cassette is provided with a
plurality of restriction sites for insertion of the nucleotide sequence
of interest to be under the transcriptional regulation of the regulatory
regions.

[0024] The expression cassette may further comprise 3' and/or 5'
untranslated region(s). By "3' untranslated region" is intended a
nucleotide sequence located downstream of a coding sequence.
Polyadenylation signal sequences and other sequences encoding regulatory
signals capable of affecting the addition of polyadenylic acid tracts to
the 3' end of the mRNA precursor are 3' untranslated regions. By "5'
untranslated region" is intended a nucleotide sequence located upstream
of a coding sequence. Other upstream or downstream untranslated elements
include enhancers. Enhancers are nucleotide sequences that act to
increase the expression of a promoter region. Enhancers are well known in
the art and include, but are not limited to, the SV40 enhancer region and
the 35S enhancer element.

[0026] The expression cassettes described herein may further comprise one
or more regulatory elements other than CTP, as well as additional CTPs
known in the art. By "regulatory element" or "regulatory region" is
intended a portion of nucleic acid found upstream or downstream of a
gene, that may be comprised of either DNA or RNA, or both DNA and RNA and
that is involved in gene expression. Regulatory elements may be capable
of mediating organ specificity, or controlling developmental or temporal
gene activation and include promoter elements, core promoter elements,
elements that are inducible in response to an external stimulus, elements
that are activated constitutively, transcriptional terminators,
polyadenylation signals, and elements that decrease or increase promoter
activity such as negative regulatory elements or transcriptional
enhancers, respectively. By "cis-acting" is intended a sequence that is
physically contiguous with the transcribed sequence. Cis-acting sequences
typically interact with proteins or other molecules to carry out (turn
on/off, regulate, modulate, etc.) transcription. By "transcriptional
enhancer" is intended a nucleic acid sequence that, when positioned
proximate to a promoter and present in a transcription medium capable of
supporting transcription, confers increased transcription activity
compared to that resulting from the promoter in the absence of the
enhancer. Enhancers may function upstream, within, or downstream of a
gene, even as far away as 50 kilobases from the transcriptional
initiation site. Enhancers may also function independently of their
orientation. By "transcriptional terminator" is intended a DNA sequence
that includes a nucleotide base pair sequence necessary for reducing or
eliminating transcription. By "polyadenylation signal" is intended a
sequence that controls the termination of transcription and translation.

[0027] In one aspect of the invention, synthetic DNA sequences are
designed for a given polypeptide, such as the chloroplast-targeted
polypeptides useful in the methods disclosed herein. Expression of the
open reading frame of the synthetic DNA sequence in a cell results in
production of the polypeptide. Synthetic DNA sequences can be useful to
simply remove unwanted restriction endonuclease recognition sites, to
facilitate DNA cloning strategies, to alter or remove any potential codon
bias, to alter or improve GC content, to remove or alter alternate
reading frames, and/or to alter or remove intron/exon splice recognition
sites, polyadenylation sites, Shine-Delgarno sequences, unwanted promoter
elements and the like that may be present in a native DNA sequence. It is
also possible that synthetic DNA sequences may be utilized to introduce
other improvements to a DNA sequence, such as introduction of an intron
sequence, creation of a DNA sequence that in expressed as a protein
fusion to organelle targeting sequences, such as chloroplast transit
peptides, apoplast/vacuolar targeting peptides, or peptide sequences that
result in retention of the resulting peptide in the endoplasmic
reticulum. Synthetic genes can also be synthesized using host
cell-preferred codons for improved expression, or may be synthesized
using codons at a host-preferred codon usage frequency. See, for example,
Campbell and Gowri (1990) Plant Physiol. 92:1-11; U.S. Pat. Nos.
6,320,100; 6,075,185; 5,380,831; and 5,436,391, U.S. Published
Application Nos. 20040005600 and 20010003849, and Murray et al. (1989)
Nucleic Acids Res. 17:477-498, herein incorporated by reference.

[0028] The nucleic acids of interest to be targeted to the chloroplast may
also be optimized for expression in the chloroplast to account for
differences in codon usage between the plant nucleus and this organelle.
In this manner, the nucleic acids of interest may be synthesized using
chloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831,
herein incorporated by reference.

Variants and Fragments

[0029] Nucleic acid molecules that are fragments of the disclosed CTP
sequences are also encompassed by the present invention. By "fragment" is
intended a portion of the CTP sequence. A fragment of a nucleotide
sequence may be biologically active and hence be capable of facilitating
the translocation of a polypeptide of interest into the chloroplast of a
plant, or it may be a fragment that can be used as a hybridization probe
or PCR primer using methods disclosed below. Assays to determine whether
such fragments have CTP activity are well known in the art.

[0030] Nucleic acid molecules that are fragments of a CTP-encoding
nucleotide sequence disclosed herein may comprise at least about 90, 100,
125, 150, 175, 200, 225, 250, 275, 300, contiguous nucleotides, or up to
the number of nucleotides present in a full-length CTP sequence disclosed
herein (for example, 306 nucleotides for SEQ ID NO:1) depending upon the
intended use. By "contiguous" nucleotides is intended nucleic acid
residues that are immediately adjacent to one another. Biologically
active fragments of the CTP-encoding sequences of the present invention
will encode a CTP that retains activity. By "retains CTP activity" is
intended that the fragment will direct the translocation into the
chloroplast of at least about 30%, at least about 50%, at least about
70%, or at least about 80% of the polypeptide encoded by the nucleotide
sequence of interest. In one embodiment, a fragment of a CTP-encoding
nucleotide sequence disclosed herein may comprise one or more deletions
of SEQ ID NO:1, 2, 4, or 6, including up to about 2, about 3, about 4,
about 5, about 6, about 7, about 8, about 9, about 10, about 12, about
15, about 18, about 21, about 24, about 27, about 30 or more deletions.
In another embodiment, a fragment of a CTP-encoding nucleotide sequence
disclosed herein may encode an amino acid comprising one or more
deletions of SEQ ID NO:3, 5, or 7, including up to about 2, about 3,
about 4, about 5, about 6, about 7, about 8, about 9, about 10 or more
amino acid deletions.

[0031] A biologically active portion of a CTP can be prepared by isolating
a portion of one of the CTP sequences of the invention and assessing the
activity of that portion of the CTP. Methods for measuring CTP activity
are well known in the art. See the section entitled "Evaluation of CTP
Activity" for examples of suitable methods.

[0032] Variants of the CTP-encoding nucleotide sequences or the CTP amino
acid sequences disclosed herein are also encompassed. By "variant" is
intended a sufficiently identical sequence, or a sequence that differs by
at least one amino acid from a native chloroplast transit peptide.
CTP-encoding sequences encompassed by the present invention are
sufficiently identical to the nucleotide sequence of SEQ ID NO:1, 2, 4,
or 6. CTP sequences encompassed herein are sufficiently identical to the
amino acid sequence of SEQ ID NO:3, 5, or 7. By "sufficiently identical"
is intended a nucleotide sequence that has at least about 70% or 75%,
about 80% or 85% sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% sequence identity compared to a reference sequence
using one of the alignment programs as described herein.

[0033] In one embodiment, the variants disclosed herein include nucleotide
or amino acid substitutions, deletions, truncations, and insertions of
one or more nucleotides of SEQ ID NO:1, 2, 4, or 6, or one or more amino
acids of SEQ ID NO:3, 5, or 7, including up to about 2, about 3, about 4,
about 5, about 6, about 7, about 8, about 9, about 10, about 15, about
20, about 25, about 30 or more amino acid substitutions, deletions or
insertions.

[0034] Naturally occurring variants can be identified with the use of
well-known molecular biology techniques, such as polymerase chain
reaction (PCR) and hybridization techniques as outlined below. Variant
nucleotide sequences also include synthetically derived nucleotide
sequences that have been generated, for example, by using site-directed
mutagenesis but which still have CTP activity as defined herein.

[0035] Variants encompassed by the present invention are biologically
active, that is they continue to possess the desired biological activity
of the native sequence, that is, retaining CTP activity (i.e.,
facilitating translocation of the expressed polypeptide to the
chloroplast). By "retains CTP activity" is intended that the variant will
direct the translocation to the chloroplast of at least about 30%, at
least about 50%, at least about 70%, or at least about 80% of the
polypeptide encoded by the nucleotide sequence of interest. Methods for
measuring CTP activity are well known in the art. See the section
entitled "Evaluation of CTP Activity" for examples of suitable methods.

[0036] The skilled artisan will further appreciate that changes to the CTP
can be introduced by mutation into the nucleotide sequence encoding the
CTPs of the invention without altering the ability of the CTP to drive
translocation of a polypeptide in the chloroplast of a plant cell. Thus,
variant isolated nucleic acid molecules can be created by introducing one
or more nucleotide substitutions, additions, or deletions into the
corresponding nucleotide sequence disclosed herein. Mutations can be
introduced by standard techniques, such as site-directed mutagenesis and
PCR-mediated mutagenesis. Such variant nucleotide sequences are also
encompassed by the present invention.

[0037] Alternatively, variant nucleotide sequences can be made by
introducing mutations randomly along all or part of the CTP sequence,
such as by saturation mutagenesis, and the resultant mutants can be
screened for ability to drive translocation of an operably linked
polypeptide sequence into the chloroplast a plant cell.

[0038] By "operably linked" is intended a functional linkage between a
regulatory element (e.g., a CTP) and a second sequence, wherein the CTP
sequence directs the translocation of the polypeptide of interest to the
chloroplast of a plant cell. Generally, but not always, operably linked
means that the nucleic acid sequences being linked are contiguous and,
where necessary to join two protein coding regions, contiguous and in the
same reading frame.

[0039] To determine the percent identity of two nucleic acids, the
sequences are aligned for optimal comparison purposes. The percent
identity between the two sequences is a function of the number of
identical positions shared by the sequences (i.e., percent identity
=number of identical positions/total number of positions (e.g.,
overlapping positions)×100). In one embodiment, the two sequences
are the same length. In another embodiment, the comparison is across the
entirety of the reference sequence (e.g., SEQ ID NO:1, 2, 4, or 6). The
percent identity between two sequences can be determined using techniques
similar to those described below, with or without allowing gaps. In
calculating percent identity, typically exact matches are counted.

[0040] The determination of percent identity between two sequences can be
accomplished using a mathematical algorithm. A nonlimiting example of a
mathematical algorithm utilized for the comparison of two sequences is
the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA
87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci.
USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN
program of Altschul et al. (1990) J. Mol. Biol. 215:403. BLAST nucleotide
searches can be performed with the BLASTN program, score=100,
wordlength=12, to obtain nucleotide sequences homologous to sequences of
the invention. To obtain gapped alignments for comparison purposes,
Gapped BLAST can be utilized as described in Altschul et al. (1997)
Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to
perform an iterated search that detects distant relationships between
molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped
BLAST, and PSI-Blast programs, the default parameters of the respective
programs (e.g., BLASTN) can be used. See, www.ncbi.nlm.nih.gov. Another
non-limiting example of a mathematical algorithm utilized for the
comparison of sequences is the ClustalW algorithm (Higgins et al. (1994)
Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences and aligns
the entirety of the DNA sequence, and thus can provide data about the
sequence conservation of the entire nucleotide sequence. The ClustalW
algorithm is used in several commercially available DNA analysis software
packages, such as the ALIGNX module of the vector NTi Program Suite
(Informax, Inc). A non-limiting example of a software program useful for
analysis of ClustalW alignments is GeneDoc®. Genedoc® (Karl
Nicholas) allows assessment of DNA similarity and identity between
multiple genes. Another preferred, non-limiting example of a mathematical
algorithm utilized for the comparison of sequences is the algorithm of
Myers and Miller (1988) CABIOS 4:11-17. Such an algorithm is incorporated
into the ALIGN program (version 2.0), which is part of the GCG sequence
alignment software package (available from Accelrys, Inc., 9865 Scranton
Rd., San Diego, Calif., USA).

[0041] Unless otherwise stated, GAP Version 10, which uses the algorithm
of Needleman and Wunsch (1970) J. Mol. Biol. 48(3):443-453, will be used
to determine sequence identity or similarity using the following
parameters: % identity and % similarity for a nucleotide sequence using
GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring
matrix; % identity or % similarity for an amino acid sequence using GAP
weight of 8 and length weight of 2, and the BLOSUM62 scoring program.
Equivalent programs may also be used. By "equivalent program" is intended
any sequence comparison program that, for any two sequences in question,
generates an alignment having identical nucleotide residue matches and an
identical percent sequence identity when compared to the corresponding
alignment generated by GAP Version 10.

[0042] Using methods such as PCR, hybridization, and the like,
corresponding sequences from other organisms, particularly other algal
organisms, can be identified, such sequences having substantial identity
to the sequences of the invention. See, for example, Sambrook J., and
Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual. (Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and Innis, et
al. (1990) PCR Protocols: A Guide to Methods and Applications (Academic
Press, NY). Sequences identified by their identity to the CTP sequences
set forth herein are encompassed by the present invention.

[0043] In another embodiment, CTPs can be identified based on the
identification of sequences known to comprise CTPs. For example,
chloroplast-targeted sequences can be identified based on similarity to
the chloroplast-targeted proteins disclosed herein or known in the art
(e.g., acetolactate synthase (AHAS), small subunit (SSU), and EPSPS). The
CTP sequence from these targeted proteins can be identified using methods
known in the art. See, for example, Emanuelsson and von Heijne (2001)
Biochimica et Biophysica Acta 1541:114-119; Nielson et al. (1997) Protein
Eng. 10:1-6; and, Nielson and Krogh (1998) Intell. Syst. Mol. Biol.
6:122-130, each of which is herein incorporated by reference in its
entirety. A variety of computer programs are also available for
identifying. See, for example, ChloroP (which can be found at the
internet address cbs.dtu.dk/services/ChloroP/); Predotar (which can be
found at the internet address inra.fr/Internet/Produits/Predotar/); and,
SignalP (which can be found at the internet address
cbs.dtu.dk/services/SignalP/).

[0044] Oligonucleotide primers can be designed for use in PCR reactions to
amplify corresponding DNA sequences from cDNA or genomic DNA from an
organism of interest. Methods for designing PCR primers and PCR cloning
are generally known in the art and are disclosed in Sambrook et al.
(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.). See also Innis et al., eds.
(1990) PCR Protocols: A Guide to Methods and Applications (Academic
Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic
Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual
(Academic Press, New York). Known methods of PCR include, but are not
limited to, methods using paired primers, nested primers, single specific
primers, degenerate primers, gene-specific primers, vector-specific
primers, and partially-mismatched primers.

[0045] In a hybridization method, all or part of a known nucleotide
sequence can be used to screen cDNA or genomic libraries. Methods for
construction of such cDNA and genomic libraries are generally known in
the art and are disclosed in Sambrook and Russell, 2001, supra. The
hybridization probes may be genomic DNA fragments, cDNA fragments, RNA
fragments, or other oligonucleotides, and may be labeled with a
detectable group such as 32P, or any other detectable marker, such
as other radioisotopes, a fluorescent compound, an enzyme, or an enzyme
co-factor. Probes for hybridization can be made by labeling synthetic
oligonucleotides based on the known CTP-encoding sequence disclosed
herein or primers to the known chloroplast targeted protein. Degenerate
primers designed on the basis of conserved nucleotides in the nucleotide
sequence can additionally be used. The probe typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions to at
least about 12, at least about 20, at least about 25, 30, 35, 40, 45, 50,
55, 60, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 consecutive
nucleotides of the CTP-encoding sequence of the invention, a nucleotide
sequence encoding a chloroplast targeted protein, or a fragment or
variant thereof. Preparation of probes for hybridization is generally
known in the art and is disclosed in Sambrook and Russell, 2001, supra,
herein incorporated by reference.

[0046] For example, the entire CTP-encoding sequence disclosed herein (or
coding sequence for chloroplast-targeted protein), or one or more
portions thereof, may be used as a probe capable of specifically
hybridizing to corresponding CTP-like sequences. To achieve specific
hybridization under a variety of conditions, such probes include
sequences that are unique and are at least about 10 nucleotides in
length, or at least about 20 nucleotides in length. Such probes may be
used to amplify corresponding CTP-encoding sequences from a chosen
organism by PCR. This technique may be used to isolate additional coding
sequences from a desired organism or as a diagnostic assay to determine
the presence of coding sequences in an organism. Hybridization techniques
include hybridization screening of plated DNA libraries (either plaques
or colonies; see, for example, Sambrook et al. (1989) Molecular Cloning:
A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.).

[0047] Hybridization of such sequences may be carried out under stringent
conditions. By "stringent conditions" or "stringent hybridization
conditions" is intended conditions under which a probe will hybridize to
its target sequence to a detectably greater degree than to other
sequences (e.g., at least 2-fold over background). Stringent conditions
are sequence-dependent and will be different in different circumstances.
By controlling the stringency of the hybridization and/or washing
conditions, target sequences that are 100% complementary to the probe can
be identified (homologous probing). Alternatively, stringency conditions
can be adjusted to allow some mismatching in sequences so that lower
degrees of similarity are detected (heterologous probing). Generally, a
probe is less than about 1000 nucleotides in length, or less than 500
nucleotides in length.

[0048] Typically, stringent conditions will be those in which the salt
concentration is less than about 1.5 M Na ion, typically about 0.01 to
1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least about 30° C. for short probes (e.g., 10 to
50 nucleotides) and at least about 60° C. for long probes (e.g.,
greater than 50 nucleotides). Stringent conditions may also be achieved
with the addition of destabilizing agents such as formamide. Exemplary
low stringency conditions include hybridization with a buffer solution of
30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at
37° C., and a wash in 1× to 2X SSC (20X SSC=3.0 M NaCl/0.3 M
trisodium citrate) at 50 to 55° C. Exemplary moderate stringency
conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1%
SDS at 37° C., and a wash in 0.5× to 1X SSC at 55 to
60° C. Exemplary high stringency conditions include hybridization
in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1X
SSC at 60 to 65° C. Optionally, wash buffers may comprise about
0.1% to about 1% SDS. Duration of hybridization is generally less than
about 24 hours, usually about 4 to about 12 hours. Optionally, wash
buffers may comprise about 0.1% to about 1% SDS.

[0049] Specificity is typically the function of post-hybridization washes,
the critical factors being the ionic strength and temperature of the
final wash solution. For DNA-DNA hybrids, the Tm can be approximated
from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:
Tm=81.5° C.+16.6 (log M)+0.41 (%GC)-0.61 (% form)-500/L;
where M is the molarity of monovalent cations, % GC is the percentage of
guanosine and cytosine nucleotides in the DNA, % form is the percentage
of formamide in the hybridization solution, and L is the length of the
hybrid in base pairs. The Tm is the temperature (under defined ionic
strength and pH) at which 50% of a complementary target sequence
hybridizes to a perfectly matched probe. Tm is reduced by about
1° C. for each 1% of mismatching; thus, Tm, hybridization,
and/or wash conditions can be adjusted to hybridize to sequences of the
desired identity. For example, if sequences with ≧90% identity are
sought, the Tm can be decreased 10° C. Generally, stringent
conditions are selected to be about 5° C. lower than the thermal
melting point (Tm) for the specific sequence and its complement at a
defined ionic strength and pH. However, severely stringent conditions can
utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower
than the thermal melting point (Tm); moderately stringent conditions
can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C.
lower than the thermal melting point (Tm); low stringency conditions
can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or
20° C. lower than the thermal melting point (Tm). Using the
equation, hybridization and wash compositions, and desired Tm, those
of ordinary skill will understand that variations in the stringency of
hybridization and/or wash solutions are inherently described. If the
desired degree of mismatching results in a Tm of less than
45° C. (aqueous solution) or 32° C. (formamide solution),
the SSC concentration can be increased so that a higher temperature can
be used. An extensive guide to the hybridization of nucleic acids is
found in Tijssen (1993) Laboratory Techniques in Biochemistry and
Molecular Biology--Hybridization with Nucleic Acid Probes, Part I,
Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current
Protocols in Molecular Biology, Chapter 2 (Greene Publishing and
Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.).

[0050] Isolated sequences that have CTP activity and which hybridize under
stringent conditions to the CTP sequences disclosed herein, or to
fragments thereof, are encompassed by the present invention.

Methods of Use

[0051] Methods of the present invention are directed to the proper
expression, translocation, and processing of chloroplast-targeted
sequences in higher plants and plant cells under the control of the CTP
sequences of the present invention. For the purposes of the present
invention, a "processed" chloroplast targeted protein is one in which the
CTP has been removed. At the time of translocation of a chloroplast
targeted protein into the chloroplast of a plant cell, the CTP is removed
from targeted protein by cleavage at a particular "cleavage site" between
the CTP and the mature protein. The cleavage site can be determined
experimentally, or may be predicted based on sequence structure (e.g., by
alignment of the unprocessed protein with chloroplast targeted proteins
in which the cleavage site is known, by analyzing the sequence for the
presence of characteristic CTP domains, and the like) or by using one or
more algorithms for cleavage site prediction as discussed elsewhere
herein (e.g., SignalP).

[0052] The transgenic plants may have a change in phenotype, including,
but not limited to, an altered pathogen or insect defense mechanism, an
increased resistance to one or more herbicides, an increased ability to
withstand stressful environmental conditions, a modified ability to
produce starch, a modified level of starch production, a modified oil
content and/or composition, a modified ability to utilize, partition
and/or store nitrogen, and the like. These results can be achieved
through the expression and targeting of a polypeptide of interest to
chloroplasts in plants, wherein the polypeptide of interest functions in
the chloroplast. The CTP sequences of the invention are useful for
targeting native sequences as well as heterologous (non-native) sequences
in higher plants. For the purposes of the present invention, "higher
plants" are considered members of the subkingdom Embryophytae. In one
embodiment, the plant is a monocotyledon. In another embodiment, the
plant is a dicotyledon.

[0053] Generally, the nucleotide sequence encoding the CTP of the
invention is provided in an expression cassette with a nucleotide
sequence of interest for expression in the plant of interest. In one
embodiment, the CTP-encoding sequences of the invention are useful for
the improved translocation of native sequences in a plant. In other
embodiments, the CTP-encoding sequences are useful for expression and
translocation of polypeptides encoded by heterologous nucleotide
sequences. By "heterologous nucleotide sequence" is intended a sequence
that is not naturally operably-linked with the CTP-encoding sequence of
the invention, including non-naturally occurring multiple copies of a
naturally occurring DNA sequence. While this nucleotide sequence is
heterologous to the CTP-encoding sequence, it may be homologous, or
"native," or heterologous, or "foreign," to the plant host. In some
cases, the transformed plant may have a change in phenotype.
"Heterologous" generally refers to the nucleic acid sequences that are
not endogenous to the cell or part of the native genome in which they are
present, and have been added to the cell by infection, transfection,
microinjection, electroporation, microprojection, or the like.

[0054] Any nucleotide sequence of interest may be used with the
CTP-encoding sequences of the invention, so long as the polypeptide
encoded by the nucleotide sequence of interest (i.e., the "polypeptide of
interest") is functional in a chloroplast. Such nucleotide sequences
include, but are not limited to, herbicide-tolerance coding sequences,
insecticidal coding sequences, nematicidal coding sequences,
antimicrobial coding sequences, antifungal coding sequences, antiviral
coding sequences, abiotic and biotic stress tolerance coding sequences,
or sequences modifying plant traits such as yield, grain quality,
nutrient content, starch quality and quantity, nitrogen fixation and/or
utilization, and oil content and/or composition. More specific genes of
interest for the present invention include, but are not limited to, genes
that improve crop yield, genes that improve desirability of crops, genes
encoding proteins conferring resistance to abiotic stress, such as
drought, temperature, salinity, toxic metals or trace elements, or those
conferring resistance to toxins such as pesticides and herbicides, or to
biotic stress, such as attacks by fungi, viruses, bacteria, insects, and
nematodes, and development of diseases associated with these organisms.
It is recognized that any gene of interest can be operably linked to the
CTP-encoding sequences of the invention and expressed in a plant, so long
as the polypeptide encoded by the gene is functional in chloroplasts.

[0055] These nucleotide sequences of interest may encode proteins involved
in providing disease or pest resistance. By "disease resistance" or "pest
resistance" is intended that the plants avoid the harmful symptoms that
are the outcome of the plant-pathogen interactions. Disease resistance
and insect resistance genes such as lysozymes or cecropins for
antibacterial protection, or proteins such as defensins, glucanases or
chitinases for antifungal protection, or Bacillus thuringiensis
endotoxins, protease inhibitors, collagenases, lectins, or glycosidases
for controlling nematodes or insects are all examples of useful gene
products. Examples of genes of interest may be found, for example, at
www.nbiap.vt.edu/cfdocs/fieldtests2.cfm.

[0057] An "herbicide resistance protein" or a protein resulting from
expression of an "herbicide resistance-encoding nucleic acid molecule"
includes proteins that confer upon a cell the ability to tolerate a
higher concentration of an herbicide than cells that do not express the
protein, or to tolerate a certain concentration of an herbicide for a
longer period of time than cells that do not express the protein.
Herbicide resistance traits may be introduced into plants by genes coding
for resistance to herbicides that act to inhibit the action of
acetolactate synthase (ALS), in particular the sulfonylurea-type
herbicides, genes coding for resistance to herbicides that act to inhibit
the action of glutamine synthase, such as phosphinothricin or basta
(e.g., the bar gene), glyphosate (e.g., the EPSP synthase gene and the
GAT gene) or other such genes known in the art.

[0058] Genes that improve crop yield include dwarfing genes, such as Rht1
and Rht2 (Peng et al. (1999) Nature 400:256-261), and those that increase
plant growth, such as ammonium-inducible glutamate dehydrogenase. Genes
that improve desirability of crops include, for example, those that allow
plants to have a reduced saturated fat content, those that boost the
nutritional value of plants, and those that increase grain protein. Genes
that improve salt tolerance are those that increase or allow plant growth
in an environment of higher salinity than the native environment of the
plant into which the salt-tolerant gene(s) has been introduced.

Plant Transformation Vectors

[0059] Typically the plant expression cassette will be inserted into a
"plant transformation vector." By "transformation vector" is intended a
DNA molecule that is necessary for efficient transformation of a cell.
Such a molecule may consist of one or more expression cassettes, and may
be organized into more than one "vector" DNA molecule. For example,
binary vectors are plant transformation vectors that utilize two
non-contiguous DNA vectors to encode all requisite cis- and trans-acting
functions for transformation of plant cells (Hellens and Mullineaux
(2000) Trends in Plant Science 5:446-451). "Vector" refers to a nucleic
acid construct designed for transfer between different host cells.
"Expression vector" refers to a vector that has the ability to
incorporate, integrate and express heterologous DNA sequences or
fragments in a foreign cell. By "introducing" is intended to present to
the organism being transformed the nucleotide construct in such a manner
that the construct gains access to the interior of at least one cell of
the organism.

[0060] This plant transformation vector may be comprised of one or more
DNA vectors needed for achieving plant transformation. For example, it is
a common practice in the art to utilize plant transformation vectors that
are comprised of more than one contiguous DNA segment. These vectors are
often referred to in the art as `binary vectors`. Binary vectors as well
as vectors with helper plasmids are most often used for
Agrobacterium-mediated transformation, where the size and complexity of
DNA segments needed to achieve efficient transformation is quite large,
and it is advantageous to separate functions onto separate DNA molecules.
Binary vectors typically contain a plasmid vector that contains the
cis-acting sequences required for T-DNA transfer (such as left border and
right border), a selectable marker that is engineered to be capable of
expression in a plant cell, and a "gene of interest" (a gene engineered
to be capable of expression in a plant cell for which generation of
transgenic plants is desired). Also present on this plasmid vector are
sequences required for bacterial replication.

[0061] The cis-acting sequences are arranged in a fashion to allow
efficient transfer into plant cells and expression therein. For example,
the selectable marker gene and the gene of interest are located between
the left and right borders. Often a second plasmid vector contains the
trans-acting factors that mediate T-DNA transfer from Agrobacterium to
plant cells. This plasmid often contains the virulence functions (Vir
genes) that allow infection of plant cells by Agrobacterium, and transfer
of DNA by cleavage at border sequences and vir-mediated DNA transfer, as
in understood in the art (Hellens and Mullineaux (2000) Trends in Plant
Science, 5:446-451). Several types of Agrobacterium strains (e.g.
LBA4404, GV3101, EHA101, EHA105, etc.) can be used for plant
transformation. The second plasmid vector is not necessary for
transforming the plants by other methods such as microprojection,
microinjection, electroporation, polyethylene glycol, etc.

Plant Transformation

[0062] Methods of the invention involve introducing a nucleotide construct
into a plant. By "introducing" is intended to present to the plant the
nucleotide construct in such a manner that the construct gains access to
the interior of a cell of the plant. The methods of the invention do not
require that a particular method for introducing a nucleotide construct
to a plant is used, only that the nucleotide construct gains access to
the interior of at least one cell of the plant. Methods for introducing
nucleotide constructs into plants are known in the art including, but not
limited to, stable transformation methods, transient transformation
methods, and virus-mediated methods.

[0063] Transformation of plant cells can be accomplished by one of several
techniques known in the art. By "plant" is intended whole plants, plant
organs (e.g., leaves, stems, roots, etc.), seeds, plant cells,
propagules, embryos and progeny of the same. Plant cells can be
differentiated or undifferentiated (e.g. callus, suspension culture
cells, protoplasts, leaf cells, root cells, phloem cells, pollen).
"Transgenic plants" or "transformed plants" or "stably transformed"
plants or cells or tissues refer to plants that have incorporated or
integrated exogenous nucleic acid sequences or DNA fragments into the
plant cell. By "stable transformation" is intended that the nucleotide
construct introduced into a plant integrates into the genome of the plant
and is capable of being inherited by progeny thereof.

[0064] In general, plant transformation methods involve transferring
heterologous DNA into target plant cells (e.g. immature or mature
embryos, suspension cultures, undifferentiated callus, protoplasts,
etc.), followed by applying a maximum threshold level of appropriate
selection (depending on the selectable marker gene) to recover the
transformed plant cells from a group of untransformed cell mass. Explants
are typically transferred to a fresh supply of the same medium and
cultured routinely. Subsequently, the transformed cells are
differentiated into shoots after placing on regeneration medium
supplemented with a maximum threshold level of selecting agent. The
shoots are then transferred to a selective rooting medium for recovering
rooted shoot or plantlet. The transgenic plantlet then grow into mature
plant and produce fertile seeds (e.g. Hiei et al. (1994) The Plant
Journal 6:271-282; Ishida et al. (1996) Nature Biotechnology 14:745-750).
A general description of the techniques and methods for generating
transgenic plants are found in Ayres and Park (1994) Critical Reviews in
Plant Science 13:219-239 and Bommineni and Jauhar (1997) Maydica
42:107-120. Since the transformed material contains many cells; both
transformed and non-transformed cells are present in any piece of
subjected target callus or tissue or group of cells. The ability to kill
non-transformed cells and allow transformed cells to proliferate results
in transformed plant cultures. Often, the ability to remove
non-transformed cells is a limitation to rapid recovery of transformed
plant cells and successful generation of transgenic plants. Molecular and
biochemical methods may be used to confirm the presence of the integrated
heterologous gene of interest in the genome of transgenic plant.

[0066] Methods for transformation of chloroplasts are known in the art.
See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA
87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA
90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. The method relies
on particle gun delivery of DNA containing a selectable marker and
targeting of the DNA to the plastid genome through homologous
recombination. Additionally, plastid transformation can be accomplished
by transactivation of a silent plastid-borne transgene by
tissue-preferred expression of a nuclear-encoded and plastid-directed RNA
polymerase. Such a system has been reported in McBride et al. (1994)
Proc. Natl. Acad. Sci. USA 91:7301-7305.

[0067] The cells that have been transformed may be grown into plants in
accordance with conventional ways. See, for example, McCormick et al.
(1986) Plant Cell Reports 5:81-84. These plants may then be grown, and
either pollinated with the same transformed strain or different strains,
and the resulting hybrid having constitutive expression of the desired
phenotypic characteristic identified. Two or more generations may be
grown to ensure that expression of the desired phenotypic characteristic
is stably maintained and inherited and then seeds harvested to ensure
expression of the desired phenotypic characteristic has been achieved. In
this manner, the present invention provides transformed seed (also
referred to as "transgenic seed") having a nucleotide construct of the
invention, for example, an expression cassette of the invention, stably
incorporated into their genome.

[0070] This invention is particularly suitable for any member of the
monocot plant family including, but not limited to, maize, rice, barley,
oats, wheat, sorghum, rye, sugarcane, pineapple, yams, onion, banana,
coconut, and dates.

Evaluation of Plant Transformation

[0071] Following introduction of heterologous foreign DNA into plant
cells, the transformation or integration of heterologous DNA in the plant
genome is confirmed by various methods such as analysis of nucleic acids
or proteins and metabolites associated with the integrated DNA.

[0072] PCR analysis is a rapid method to screen transformed cells, tissue
or shoots for the presence of incorporated DNA at the earlier stage
before transplanting into the soil (Sambrook and Russell, 2001. Molecular
Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.). PCR is carried out using oligonucleotide primers
specific to the gene of interest or Agrobacterium vector background, etc.

[0073] Plant transformation may be confirmed by Southern blot analysis of
genomic DNA (Sambrook and Russell, 2001, supra). In general, total DNA is
extracted from the transformant, digested with appropriate restriction
enzymes, fractionated in an agarose gel and transferred to a
nitrocellulose or nylon membrane. The membrane or "blot" then is probed
with, for example, radiolabeled 32P target DNA fragment to confirm
the integration of introduced DNA in the plant genome according to
standard techniques (Sambrook and Russell, 2001, supra).

[0074] In Northern blot analysis, RNA is isolated from specific tissues of
transformant, fractionated in a formaldehyde agarose gel, blotted onto a
nylon filter according to standard procedures that are routinely used in
the art (Sambrook and Russell, 2001, supra). Expression of RNA encoded by
a heterologous gene operably linked to the CTP-encoding sequence is then
tested by hybridizing the filter to a radioactive probe derived from the
heterologous gene, by methods known in the art (Sambrook and Russell,
2001, supra).

Evaluation of CTP Activity

[0075] Assays to determine the efficiency by which the CTP sequences of
the invention target a protein of interest to a chloroplast are known.
See, for example, Mishkind et al. (1985) J of Cell Biol 100:226-234,
which is herein incorporated by reference in its entirety. A reporter
gene such as-glucuronidase (GUS), chloramphenicol acetyl transferase
(CAT), or green fluorescent protein (GFP) is operably linked to the CTP
sequence. This fusion is placed behind the control of a suitable
promoter, ligated into a transformation vector, and transformed into a
plant or plant cell. Following an adequate period of time for expression
and localization into the chloroplast, the chloroplast fraction is
extracted and reporter activity assayed. The ability of the isolated
sequences to target and deliver the reporter protein to the chloroplast
can be compared to other known CTP sequences. See de Castro Silva Filho
et al. (1996) Plant Mol. Biol. 30: 769-780. Protein import can also be
verified in vitro through the addition of proteases to the isolated
chloroplast fraction. Proteins which were successfully imported into the
chloroplast are resistant to the externally added proteases whereas
proteins that remain in the cytosol are susceptible to digestion. Protein
import can also be verified by the presence of functional protein in the
chloroplast using standard molecular techniques for detection, or by
evaluating the phenotype resulting from expression of a chloroplast
targeted protein.

[0076] The following examples are offered by way of illustration and not
by way of limitation.

EXPERIMENTAL

Example 1

Chlamydomonas AHAS CTP

[0077] The Chlamydomonas AHAS CTP (SEQ ID NO:3) was identified from
comparison of the full-length Chlamydomonas AHAS ("ChlamyAHAS") peptide
(GENBANK accession AF022816; SEQ ID NO:8) with several AHAS peptides from
plants, bacteria, yeast and fungi. This alignment shows that the
ChlamyAHAS CTP shows very little sequence conservation with plant CTPs,
whereas the mature proteins show conserved elements. Thus, by comparing
the multiple AHAS proteins with the ChlamyAHAS protein, it was inferred
that amino acids 1-99 of the Chlamydomonas AHAS protein comprised the
CTP. The predicted processing site for this CTP upon import into the
Chlamydomonas chloroplast is at approximately amino acid 92 of SEQ ID
NO:8.

Example 2

Chlamydomonas RuBisCo Small Subunit (SSU) CTP

[0078] The Chlamydomonas SSU CTP (SEQ ID NO:5) was identified from
comparison of the full-length Chlamydomonas SSU ("ChlamySSU") peptide
(GENBANK accession CAA28160; SEQ ID NO:9) with several plant RuBisCo
small subunit peptides. This alignment shows that the ChlamySSU CTP shows
very little sequence conservation with plant CTPs, whereas the mature
proteins show conserved elements. Thus, by comparing the known processing
sites of several plant RuBisCo proteins with the ChlamySSU protein, it
was inferred that amino acids 1-45 of the Chlamydomonas SSU protein (SEQ
ID NO:9) comprised the CTP.

[0079] The cleavage site for the Chlamydomonas RuBisCO small subunit
precursor ("ChlamySSU") has been determined empirically to be between
residues 44 and 45 of SEQ ID NO:9 (Schmidt et al 1979, Journal of Cell
Biology 83:615-662).

Example 3

Chlamydomonas EPSPS CTP

[0080] The Chlamydomonas EPSPS CTP (SEQ ID NO:7) was identified from
comparison of the full-length Chlamydomonas EPSPS peptide (GENBANK
accession XP--001702942; SEQ ID NO:10) with several plant and
bacterial EPSPSs. This alignment showed that the ChlamyEPSPS CTP shows
very little sequence conservation with plant CTPs, whereas the mature
proteins show conserved elements. Thus, by comparing several EPSPS
proteins with the ChlamyEPSPS protein, it was inferred that amino acids
1-75 of the Chlamydomonas EPSPS protein (SEQ ID NO:10) comprised the CTP.
The predicted processing site for this CTP upon import into the
Chlamydomonas chloroplast is at approximately amino acid 61 of SEQ ID
NO:10.

[0081] A DNA element utilizing the Chlamydomonas CTP, including the
Chlamydomonas AHAS, Chlamydomonas SSU, or Chlamydomonas EPSPS CTPs to
generate multiple constructs for targeting of proteins to chloroplasts
can involve inclusion of convenient restriction endonuclease recognition
sites (for example a Bam HI restriction site) as well as small peptide
linkers between the chloroplast CTP and the protein (for example a
Gly-Ser-Gly tripeptide CTP; SEQ ID NO:18). Furthermore, such DNA elements
can be designed and made synthetically, in a way that the DNA sequence is
varied from the original DNA, but encodes the identical peptide.

[0082] Alternatively, one can design DNA constructs such that no
restriction enzyme sites are needed, and the CTP/protein fusion can be
accomplished by total synthesis of the combined coding region, or by PCR
based strategies, including "sewing PCR" and the like.

[0083] One can also design the CTP/protein fusion in a manner where some
of either protein is truncated. For example, one can remove one or more
amino acids from the N-terminus of a bacterially expressed protein, and
still achieve a functional fusion to a Chlamydomonas CTP.

[0084] A cassette containing a synthetically designed DNA sequence
encoding the Chlamydomonas AHAS CTP that incorporates a BamHI restriction
site and a Gly-Ser-Gly linker was designed. These DNA constructs contain
(from 5' to 3') (1) a Pst I cloning site, (2) the bases ACC to provide
"Kozak" context for efficient translation, (3) the portion of the gene
encoding the amino terminal methionine through the known transit peptide
cleavage site of the ChlamyAHAS, and including a small DNA region
encoding the amino acids C-terminal to the cleavage site, (4) DNA bases
encoding the residues Gly-Ser-Gly with an embedded BamH I cloning site,
and, (5) the coding region of the gene of interest.

[0085] A cassette containing a synthetically designed DNA sequence
encoding the Chlamydomonas SSU CTP that incorporates a BamHI restriction
site and a Gly-Ser-Gly linker was designed. These DNA constructs contain
(from 5' to 3') (1) a Pst I cloning site, (2) the bases ACC to provide
"Kozak" context for efficient translation, (3) the portion of the gene
encoding the amino terminal methionine through the known transit peptide
cleavage site of the ChlamySSU, and including a small DNA region encoding
the amino acids C-terminal to the cleavage site, (4) DNA bases encoding
the residues Gly-Ser-Gly with an embedded BamH I cloning site, and, (5)
the coding region of the gene of interest.

[0086] A cassette containing a synthetically designed DNA sequence
encoding the Chlamydomonas EPSPS CTP that incorporates a BamHI
restriction site and a Gly-Ser-Gly linker was designed. These DNA
constructs contain (from 5' to 3') (1) a Pst I cloning site, (2) the
bases ACC to provide "Kozak" context for efficient translation, (3) the
portion of the gene encoding the amino terminal methionine through the
known transit peptide cleavage site of the ChlamyEPSPS, and including a
small DNA region encoding the amino acids C-terminal to the cleavage
site, (4) DNA bases encoding the residues Gly-Ser-Gly with an embedded
BamH I cloning site, and, (5) the coding region of the gene of interest.

Example 5

Fusion of a Transit Peptide from a Non-Plant species to a Heterologous
Protein, and Proper Localization and Cleavage in Monocots: Chlamydomonas
AHAS CTP Functions in Monocots

[0087] DNA constructs were designed such that the resulting protein
encoded the Chlamydomonas AHAS transit peptide ("ChlamyAHAS") at the
N-terminus, followed by a protein fusion to a gene conferring herbicide
resistance upon cells (GRG-1; U.S. Pat. No. 7,405,347). For the
ChlamyAHAS precursor, the transit peptide cleavage sites were inferred
from alignments of the protein sequences to ALS proteins from bacteria,
fungi and yeast.

[0088] These DNA constructs contain (from 5' to 3') a Pst I cloning site,
(2) the bases ACC to provide "Kozak" context for efficient translation,
(3) the portion of the gene encoding the amino terminal methionine
through the known transit peptide cleavage site of the ChlamyAHAS CTP and
including a small DNA region encoding the amino acids C-terminal to the
cleavage site, (4) DNA bases encoding the residues Gly-Ser-Gly with an
embedded BamH I cloning site, and, (5) the coding region of the gene of
interest (in this case GRG-1).

[0089] These DNAs molecules were made synthetically (DNA 2.0 of Menlo
Park, Calif.). The DNA sequence of the region containing this construct
is provided as SEQ ID NO:11, and the resulting amino acid sequence is
provided as SEQ ID NO:12.

[0090] A control construct (pAG250) was made, which contains GRG-1
expressed from the TrpPro5 promoter, wherein the GRG-1 protein does not
have a chloroplast CTP.

[0091] This no CTP/GRG-1 construct was engineered into a vector for use in
Agrobacterium-mediated transformation of maize embryos, and transgenic
maize plants containing this construct were generated. To transformed
plants were analyzed by PCR to confirm presence of the construct in the
maize lines, and these T0 plants were then out-crossed to a
non-transgenic line to generate hemizygous T1 progeny. The resulting
T1 transgenic plants produce large amounts of GRG-1 protein.
Nonetheless, plants transformed with pAG250 and expressing unlocalized
GRG-1 are not resistant to glyphosate.

[0092] The algal CTP ChlamyAHAS /GRG-1 construct was engineered into a
vector for use in Agrobacterium-mediated transformation of maize embryos,
and transgenic maize plants containing this construct were generated.

[0093] To transformed plants were analyzed by PCR to confirm presence of
the construct in the maize lines, and these T0 plants were then
out-crossed to a non-transgenic line to generate hemizygous T1
progeny. The resulting T1 transgenic plants are resistant to spray
applications of glyphosate (as compared to non-transgenic controls).

[0094] Western blots of leaf tissue from transgenic maize plants show that
these plants express the GRG-1 protein. Furthermore, the size of the
protein identified by Western blot is consistent with import of the
protein into chloroplasts, and processing of the ChlamyAHAS/GRG-1 protein
at or near the cleavage site.

[0095] Thus the ChlamyAHAS CTP is sufficient to target GRG-1 to the maize
chloroplast, and result in a phenotype (herbicide resistance) that is not
conferred by GRG-1 in the absence of targeting to the chloroplast.

Example 6

Fusion of a Transit Peptide from a Non-Plant Species to a Heterologous
Protein, and Proper Localization and Cleavage in Monocots: Chlamsdomonas
SSU CTP Functions in Monocots

[0096] To test if an algal chloroplast CTP can function in monocots,
transgenic monocot plants were generated and expression and cleavage of
an algal CTP was assessed by Western blot analysis.

[0097] DNA constructs were designed such that the resulting protein
encoded the algal transit peptide at the N-terminus, fused to a protein
conferring herbicide resistance upon cells (in this case the GRG-8
protein; U.S. Patent Publication No. 20060150270)

[0098] These DNA constructs contain (from 5' to 3') (1) a Pst I cloning
site, (2) the bases ACC to provide "Kozak" context for efficient
translation, (3) the portion of the gene encoding the amino terminal
methionine through the known transit peptide cleavage site of the
ChlamySSU, and including a small DNA region encoding the amino acids
C-terminal to the cleavage site, (4) DNA bases encoding the residues
Gly-Ser-Gly with an embedded BamH I cloning site, and, (5) the coding
region of the gene of interest (in this case GRG-8).

[0099] The DNA sequence of the region containing this construct is
provided as SEQ ID NO:13, and the resulting amino acid sequence is
provided as SEQ ID NO:14.

[0100] This CTP/GRG-8 construct (pAG1675) was engineered into a vector for
use in Agrobacterium-mediated transformation of maize embryos, and
transgenic maize plants generated and identified.

[0101] To plants transformed with pAG1675 were analyzed by PCR to confirm
presence of the construct in the maize lines, and these T0 plants
were then out-crossed to a non-transgenic line to generate hemizygous
T1 progeny. T1 transgenic plants exhibited resistance to spray
applications of glyphosate compared to non-transgenic controls.

[0102] Western blots of leaf tissue from transgenic maize plants were
found to express the CTP/GRG-8 protein. Total leaf protein was extracted
from maize leaves (Pierce P-PER protein extract buffer) and separated on
a 4-12% Bis-Tris gel. GRG-8 protein was visualized using goat anti-GRG8
polyclonal antibodies. A non-transgenic maize extract was compared
alongside (lane 3). To evaluate CTP processing, a HIS-tagged GRG-8
protein standard was purified from an E. coli strain. The size of the
protein identified by Western blot is consistent with import of the
protein into chloroplasts, and processing of the CTP/GRG-8 protein within
the ChlamySSU CTP, at or near the predicted cleavage site.

[0103] The glyphosate spray tolerance of a transgenic maize event
expressing the Chlamydomonas SSU-GRG8 protein was compared to several
non-transgenic T0 control plants. Individual plants were transferred
to the greenhouse and grown in flats for 10 days. After 10 days, a
glyphosate concentration that approximated a 1× field spray rate (7
mM supplemented with 0.1% Tween 20 as surfactant) was applied to the
flats. The glyphosate was applied using a spray table to allow consistent
application of the herbicide to individual plants. Plants were rated
after 3 weeks to determine if the plants tolerated the glyphosate spray
(mostly green leaf material: <50% damage) or did not tolerate the
glyphosate spray (>75% damage, or plant death). The transgenic plant
showed tolerance to glyphosate, whereas each of the control plants failed
to show tolerance.

Example 9

Fusion of a Transit Peptide from a Non-Plant Species to a Heterologous
Protein, and Proper Localization and Cleavage in Monocots: Chlamydomonas
EPSPS CTP Functions in Monocots

[0104] DNA and amino acid sequences for the Chlamydomonas EPSPS precursor
were obtained from public databases. The transit peptide cleavage site
was predicted based on alignments of the protein sequences with EPSPS
proteins from bacteria, fungi and yeast. A synthetic gene was constructed
which encoded the CTP from the amino terminal methionine through the
predicted cleavage site. This DNA was ligated to create an in-frame
fusion with the start codon of a synthetic GRG-23(ace3)(R173K) gene (U.S.
Patent Application Publication No. 20080127372). This
CTP-GRG-23(ace3)(R173K) cassette was then ligated into a plant
transformation vector.

[0105] DNA constructs were designed such that the resulting protein
encoded the Chlamydomonas EPSPS ("ChlamyEPSPS") transit peptide at the
N-terminus, followed by fusion to a protein conferring herbicide
resistance upon cells (GRG-23(ace3)(R173K)).

[0106] These DNAs molecules were made synthetically (DNA 2.0 of Menlo
Park, Calif.). The DNA sequence of the region containing this construct
is provided as SEQ ID NO:15, and the resulting amino acid sequence is
provided as SEQ ID NO:16.

[0107] These DNA constructs contain (from 5' to 3'), (1) a Pst I cloning
site, (2) the bases ACC to provide "Kozak" context for efficient
translation, (3) the portion of the gene encoding the amino terminal
methionine through the known transit peptide cleavage site of the
ChlamyEPSPS and including a small DNA region encoding the amino acids
C-terminal to the cleavage site, (4) the coding region of the gene of
interest (in this case GRG-23(ace3)(R173K).

[0108] This Chlamydomonas EPSPS CTP/GRG-23(ace3)(R173K) construct was
engineered into a vector for use in Agrobacterium-mediated transformation
of maize embryos, and transgenic events identified.

[0109] To transformed plants were analyzed by PCR to confirm presence of
the construct in the maize lines, and these T0 plants were then
out-crossed to a non-transgenic line to generate hemizygous T1
progeny. The resulting T1 transgenic plants are resistant to spray
applications of glyphosate (as compared to non-transgenic controls).
Western blots of leaf tissue from transgenic maize plants were found to
express the Chlamydomonas EPSPS CTP/GRG-23(ace3)(R173K) protein. The
protein detected in plant tissues is smaller than the full-length
Chlamydomonas EPSPS--GRG-23(ace3)(R173K) protein, and is similar in size
to the native GRG-23(ace3)(R173K) protein (FIG. 2). The ability of the
Chlamydomonas EPSPS CTP/GRG-23(ace3)(R173K) protein to confer herbicide
resistance and the size of the resulting mature GRG-23(ace3)(R173K)
protein in herbicide resistant plants are both consistent with import of
the protein into chloroplasts, and processing of the Chlamydomonas EPSPS
CTP/GRG-23(ace3)(R173K) protein at or near the cleavage site.

[0110] From a Western blot of transgenic maize lines expressing
Chlamydomonas EPSPS--GRG-23(ace3)(R173K) protein, the distance migration
of protein molecular weight standards was graphed to generate a linear
plot of Log Molecular Weight vs. Distance Migration (FIG. 3). The linear
regression equation of this plot was used to calculate the apparent
molecular weight of the GRG23(ace3)(R173K) protein standard (FIG. 2, lane
8) and of the processed Chlamydomonas EPSPS--GRG-23(ace3)(R173K) protein
detected in plant extract. By this method, the apparent molecular weight
of the purified GRG23(ace3)(R173K) protein was determined to be 45,347
grams/mole, and the apparent molecular weight of the processed
Chlamydomonas EPSPS--GRG-23(ace3)(R173K) (lane 6, distance migration=48.5
mm) was determined as 46,227 grams/mole. The estimated molecular weights
of the processed proteins are consistent with processing of the
Chlamydomonas CTP several amino acids upstream of its junction with
GRG-23(ace3)(R173K) (the molecular weight of GRG23(ace3)(R173K) is
estimated to be 45,570 grams/mole). No protein of a size consistent with
unprocessed Chlamydomonas EPSPS--GRG-23(ace3)(R173K) protein (MW=52,012
grams/mole) is detected by Western blot. Therefore, the Chlamydomonas
EPSPS CTP is processed in maize at a discrete recognition site within the
Chlamydomonas CTP. Purification and N-terminal amino acid analysis of
this protein by methods known in the art would allow unambiguous
determination of the exact cleavage site within the Chlamydomonas CTP.

[0112] Approximately twelve micrograms of each purified plasmid was used
in polyethylene glycol-mediated tobacco protoplast transformation
experiments. After transformation, the protoplasts were incubated in a
growth chamber at 25° C. for 23 hours. Expression and localization
of TagGFP protein following transient expression was monitored under an
inverted fluorescent microscope.

[0113] The construct expressing the TagGFP without a chloroplast CTP was
detected only in the cytoplasm of protoplasts. However, the Chlamydomonas
AHAS CTP construct correctly delivered TagGFP into the chloroplast
resulting in accumulation of florescence in the chloroplast of these
protoplasts (FIG. 1).

Example 12

Evaluation of an Algal CTP Sequence in Soybean Cells

[0114] To assess the ability of algal chloroplast CTPs to function in
soybean cells, the ChlamyAHAS CTP construct (pAX3517), the ChlamyEPSPS
construct (pAX4562), and a control construct (pAX3521) containing the
TagGFP gene without a chloroplast transit peptide were used in
polyethylene glycol-mediated transformation of soybean protoplasts.
Expression and localization of TagGFP protein following transient
expression was monitored under an inverted fluorescent microscope.

[0115] For the control construct lacking a chloroplast CTP, TagGFP
fluorescence was observed only in the cytoplasm. Similarly, no expression
in the chloroplasts was observed from two independent constructs
expressing TagGFP with the ChlamyEPSPS chloroplast CTP. However, TagGFP
was detected in the chloroplast of protoplasts that had been transformed
with the ChlamyAHAS chloroplast CTP construct. Thus, this CTP functions
in soybean and well as tobacco cells.

[0116] All publications and patent applications mentioned in the
specification are indicative of the level of skill of those skilled in
the art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same extent as
if each individual publication or patent application was specifically and
individually indicated to be incorporated by reference.

[0117] Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of
understanding, it will be obvious that certain changes and modifications
may be practiced within the scope of the appended claims.